Broadly, gene therapy seeks to transfer new genetic material to the cells of a patient with resulting therapeutic benefit to the patient. Such benefits include treatment or prophylaxis of a broad range of diseases, disorders and other conditions.
Ex vivo gene therapy approaches involve modification of isolated cells, which are then infused, grafted or otherwise transplanted into the patient. See, e.g., U.S. Pat. Nos. 4,868,116, 5,399,346 and 5,460,959. In vivo gene therapy seeks to directly target host patient tissue in vivo.
Viruses useful as gene transfer vectors include papovavirus, adenovirus, vaccinia virus, adeno-associated virus, herpesvirus, and retroviruses.
Retroviral vectors are the vectors most commonly used in human clinical trials, since they carry a larger genetic payload than other viral vectors and they have the ability to infect cells and have their genetic material stably integrated into the host cell with high efficiency. See, e.g., WO 95/30761; WO 95/24929. Retroviruses require at least one round of target cell proliferation for transfer and integration of exogenous nucleic acid sequences into the patient. Retroviral vectors integrate randomly into the patient's genome.
Two classes of retroviral particles have been described; ecotropic, which can infect murine cells efficiently, and amphotropic, which can infect cells of many species. Their ability to integrate only into the genome of dividing cells has made retroviruses attractive for marking cell lineages in developmental studies and for delivering therapeutic or suicide genes to cancers or tumors. These vectors may be particularly useful in the central nervous system, where there is a relative lack of cell division in adult patients.
Retroviruses consist of a protein envelope that surrounds core proteins and RNA. The RNA encodes two long terminal repeats (LTRs), which include promoter and enhancer regions flanking the genome, transcriptional regulatory signals including the CAP site and polyadenylation signals, and structural genes including the env gene (encoding the envelope proteins), the gag gene (encoding the viral core proteins), and the pol gene (encoding the reverse transcriptase), as well as the packaging signal, psi (.psi.).
For use in human patients, the retroviral vectors must be replication defective. This prevents further generation of infectious retroviral particles in the target tissue--instead the replication defective vector becomes a "captive" transgene stable incorporated into the target cell genome. Typically in replication defective vectors, the gag, env, and pol genes have been deleted (along with most of the rest of the viral genome). Heterologous DNA is inserted in place of the deleted viral genes. The heterologous genes may be under the control of the endogenous heterologous promoter, another heterologous promoter active in the target cell, or the retroviral 5' LTR (the viral LTR is active in diverse tissues). Typically, retroviral vectors have a transgene capacity of about 7-8 kb.
Replication defective retroviral vectors require provision of the viral proteins necessary for replication and assembly in trans, from, e.g., engineered packaging cell lines. It is important that the packaging cells do not release replication competent virus and/or helper virus. This has been achieved by expressing viral proteins from RNAs lacking the .psi. signal, and more recently, expressing the gag/pol genes and the env gene from separate transcriptional units. In addition, in some packaging cell lines, the LTR's have been replaced with non-viral promoters and polyadenylation signals. These designs minimize the possibility of recombination leading to production of replication competent vectors, or helper viruses. See, e.g., U.S. Pat. No. 4,861,719, herein incorporated by reference.
Current gene therapy approaches are unsatisfactory. First, direct introduction of retroviral vectors into the host, by injection, or via liposome delivery, has several limitations. Because of their low titer and instability, a single infusion of viral vectors may be insufficient to result in infection (and gene transfer) to 100% of the desired host target tissue.
Second, use of an externalized shunt or tube is not optimal because the externalized tube or shunt could lead to infections in the patient, particularly in the brain if the treatment site is in the central nervous system ("CNS"). In addition, this is a very difficult approach for delivery to the brain parenchyma.
Third, current methods of in vivo gene therapy do not readily allow termination of, or adjustments to, the gene therapy protocol once the viral vectors have been introduced into the patient.
Certain gene therapy protocols have called for grafting of retrovirus packaging cell lines into the rodent brain for the treatment of glioblastoma. The packaging cells then release replication-incompetent retroviral particles containing genes that are desirably transferred to the tumor cells. See, e.g., Takamiya et al., J. Neurosurg., 79, pp. 104-110 (1993); Short et al., J. Neuroscience Res., 27, pp. 427-33 (1990). This method too has several limitations in human patients.
First, the injection of 10.sup.9 (or less) xenogeneic (or even allogeneic) packaging cells can create a severe immune reaction, requiring immunosuppression of the patient.
Second, a single "graft" of virus-producing cells may not be sufficient to provide the necessary number of viral particles over a biologically significant time course to kill the targeted tumor cells. The grafted packaging cells are likely to be rejected by the patient's immune system within about 7 days. Vector production may only occur for a significantly shorter period of time. It would be unlikely that 100% of the tumor cells would be infected after this limited duration. In addition, the severe immune reaction that is likely to result from the first injection may preclude subsequent injection(s) of cells that may be necessary for complete treatment. The potential reaction to such subsequent injections could prove to be dangerous or fatal.
Third, there is no mechanism in current protocols to prevent unencapsulated packaging cells from migrating in situ. The inability to retain implanted cells in a fixed location may make "naked" packaging cells unsuitable as therapeutic agents because of the possibility that the packaging cells themselves could be tumorigenic. Implantation of naked packaging cell lines is particularly a problem in immunosuppressed patients, because in those patients the immune system has a reduced ability to destroy the packaging cells should they become tumorigenic. Further, the ability of naked packaging cells to migrate increases the chances of inadvertent infection of dividing host tissues in various regions outside the CNS.
Fourth, because the grafted packaging cells in the patient are not well isolated from the patient's own tissue, they cannot be readily retrieved or manipulated, and thus do not allow termination of, or adjustments to, the gene therapy protocol once the cells are implanted.
It would be useful to provide devices and methods of gene therapy for the localized in vivo continuous delivery of viral vector particles, while preventing or reducing in situ migration of the viral vectors or packaging cells. It is also desirable to provide devices and methods of gene therapy with a reduced likelihood of eliciting an immune response in the patient. It is further desirable to provide devices and methods that permit a repeatable therapy, that can be easily and rapidly adjusted or terminated.